Abstract
The proteasome is an intracellular protease complex consisting of the 20S catalytic core and its associated regulators, including the 19S complex, PA28αβ, PA28γ, PA200, and PI31. Inhibition of the proteasome induces autoregulatory de novo formation of 20S and 26S proteasome complexes. Formation of alternative proteasome complexes, however, has not been investigated so far. We here show that catalytic proteasome inhibition results in fast recruitment of PA28γ and PA200 to 20S and 26S proteasomes within 2-6 h. Rapid formation of alternative proteasome complexes did not involve transcriptional activation of PA28γ and PA200 but rather recruitment of preexisting activators to 20S and 26S proteasome complexes. Recruitment of proteasomal activators depended on the extent of active site inhibition of the proteasome with inhibition of β5 active sites being sufficient for inducing recruitment. Moreover, specific inhibition of 26S proteasome activity via siRNA-mediated knockdown of the 19S subunit RPN6 induced recruitment of only PA200 to 20S proteasomes, whereas PA28γ was not mobilized. Here, formation of alternative PA200 complexes involved transcriptional activation of the activator. Alternative proteasome complexes persisted when cells had regained proteasome activity after pulse exposure to proteasome inhibitors. Knockdown of PA28γ sensitized cells to proteasome inhibitor-mediated growth arrest. Thus, formation of alternative proteasome complexes appears to be a formerly unrecognized but integral part of the cellular response to impaired proteasome function and altered proteostasis.
Highlights
The proteasome, one of the main proteolytic systems of the cell, is essential for maintenance of protein homeostasis and of cellular functions
The proteasome activator 28 (PA28) family, which comprises the heptameric PA28␣/ and PA28␥ complexes, and the proteasome activator 200 (PA200), function in an ubiquitin- and ATP-independent manner unless they assemble into so-called hybrid proteasome complexes that contain one 19S regulator attached to one side of the 20S core (3, 6 –9)
Catalytic Proteasome Inhibition Induces Formation of Alternative Proteasome Complexes—Primary human lung fibroblasts (phLF) were treated with a single dose of the proteasome inhibitor bortezomib (BZ, 10 nM) for 24 h and formation of proteasome complexes was analyzed via native gel electrophoresis
Summary
The proteasome, one of the main proteolytic systems of the cell, is essential for maintenance of protein homeostasis and of cellular functions. In-gel activity assays with a specific substrate for the chymotrypsin-like (CT-L) activity demonstrated effective inhibition of 20S and 26S/30S proteasome complexes (Fig. 1). In solvent-treated control cells, PA28␥ mainly formed free multimers, which were recruited to 20S and 26S complexes in response to proteasome inhibition.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.